Abstract 4364758: A Novel tRNA-Derived Small RNA, tRF-1:32-Glu-TTC-2-M2, Regulates Right Ventricular Remodeling and Fibrosis by Targeting P4HA2 in Pulmonary Arterial Hypertension

Background: Right ventricular (RV) function is the principal determinant of exercise capacity and survival in patients with pulmonary arterial hypertension (PAH). Despite its clinical significance, the molecular mechanisms driving maladaptive RV remodeling and fibrosis remain incompletely understood, limiting therapeutic options and biomarker discovery. Emerging evidence highlights the regulatory roles of tRNA-derived small RNAs (tsRNAs) in cardiovascular disease, yet their involvement in PAH-associated RV fibrosis has not been elucidated. Methods: We performed high-throughput sequencing to profile tsRNA expression in plasma from PAH patients and RV tissues from monocrotaline (MCT) rats and established two rat models of PAH (MCT and SU5416 combined with hypoxia). Functional effects of tRF-1:32-Glu-TTC-2-M2, a significantly downregulated tsRNA, were assessed by AAV9-mediated overexpression in vivo and mimic transfection in primary cardiac fibroblasts in vitro. Right heart function was evaluated by echocardiography and cardiac magnetic resonance imaging. Protein interactions were investigated by chromatin isolation by RNA purification coupled with mass spectrometry, RNA pull-down, and RNA immunoprecipitation assays. Rescue experiments validated downstream targeted protein. Results: tRF-1:32-Glu-TTC-2-M2 was markedly reduced in the RV but not lung tissues of PAH patients and rat models, inversely correlating with RV fibrosis and dysfunction. Overexpression of tRF-1:32-Glu-TTC-2-M2 significantly ameliorated RV fibrosis, improved RV function, and enhanced survival independently of pulmonary vascular remodeling. Mechanistically, tRF-1:32-Glu-TTC-2-M2 directly interacted with prolyl 4-hydroxylase subunit alpha-2 (P4HA2), inhibiting its protein expression and enzymatic activity, resulting in decreased collagen synthesis and cardiac fibroblast activation. Restoration of P4HA2 partially reversed these antifibrotic effects. Furthermore, plasma levels of tRF-1:32-Glu-TTC-2-M2 correlated with clinical indices of RV function, highlighting its potential as a diagnostic and prognostic biomarker. Conclusions: Our findings reveal a novel tsRNA-mediated regulatory axis in PAH-associated RV fibrosis, where tRF-1:32-Glu-TTC-2-M2 attenuates maladaptive remodeling through inhibition of P4HA2. This study identifies tRF-1:32-Glu-TTC-2-M2 as both a promising therapeutic target and biomarker for PAH, paving the way for RV-directed precision medicine strategies.